Purification of aldose reductase from human placenta and stabilization of the inhibitor binding site

Abstract Aldose reductase from human placenta was purified to homogeneity by a rapid (2 day) and efficient purification scheme involving Red Sepharose affinity chromatography, chromatofocusing and high performance liquid chromatography on a size-exclusion column. Addition of NADP + at all steps in the purification of aldose reductase and during storage of the enzyme at −20 stabilized both the enzyme active site and the major site for binding of aldose reductase inhibitors such as sorbinil and tolrestat. Aldose reductase is a monomer with a molecular mass of 38 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apparent pI 5.9. Placenta aldose reductase exhibited no crossreactivity with aldehyde reductase from human liver in an ELISA assay specificity for aldehydes, was specific for NADPH, and was activated by sulfate.